PROJECT SUMMARY/ABSTRACT Lyme disease is caused by infection with the spirochete Borrelia burgdorferi. B. burgdorferi is maintained in nature in an enzootic cycle comprising a tick vector and vertebrate host. The spirochete shifts its source of carbon and energy from glucose in the vertebrate to glycerol in the tick: utilization of glycerol is a crucial adaptation for the spirochete to persist in the tick and successfully transmit to a vertebrate. We hypothesize that the interface of glycerol metabolism and glycolysis, linked by interconversion of glycerol-3-phosphate and dihydroxyacetone phosphate, is the metabolic linchpin for navigating the enzootic cycle. The central hypothesis of this application is that B. burgdorferi post-transcriptionally regulates expression of the glycerol metabolism (glp) operon to utilize glycerol in order to survive in its tick vector. We endeavor to dissect the molecular mechanisms regulating expression of the glp operon, including the specific roles of a novel antisense RNA and the cyclic-dimeric GMP (c-di-GMP)-binding protein PlzA. The following specific aims are proposed toward achieving this objective: 1) define the role of the genes involved in glycerol metabolism during the enzootic cycle; 2) determine the molecular mechanism of glp operon regulation; and 3) define the function of PlzA and determine its role in regulating the glp operon. Genetic, biochemical, molecular, and transcriptomic approaches, as well as the tick-mouse model of Lyme disease, will be utilized to experimentally test these hypotheses. In this innovative application, the Multiple-PIs are in the unique position of having the experience, expertise, and experimental models to enhance the mechanistic understanding of carbohydrate metabolism and its regulation during the persistence of the Lyme disease agent in its tick vector. The long-term objective of this proposal is to understand the regulatory mechanisms that allow B. burgdorferi to respond and adapt to varied carbon sources in the tick-to-mammal transmission and in Lyme disease pathogenesis, which will lead to improved diagnostic, prevention, and treatment strategies because glycerol and glucose utilization are critical for persistence during tick and vertebrate infection, respectively; this is relevant to the mission of the agency to ?seek fundamental knowledge? for the sake of alleviating human disease.